Our recent efforts have examined novel activating Ly-49 NK receptors. Murine Ly-49D augments NK cell function upon recognition of target cells expressing H-2Dd. In addition, we demonstrate that Ly-49D receptor ligation can lead to the rapid and potent secretion of IFN-g. Cytokine secretion can be induced from Ly-49D+ NK cells after receptor ligation with Ab or after interaction with target cells expressing their H-2Dd ligand. Consistent with the dominant inhibitory function of Ly-49G, NK cells coexpressing Ly-49D and Ly-49G show a profound reduction in IFN-g secretion after interaction with targets expressing their common ligand, H-2Dd. It is well known that NK cells expressing both receptors are presented with a ligand, inhibition dominates the functional outcome. We have demonstrated that costimulation of the activating Ly-49D murine NK cell receptor with IL-12 or IL-18 is capable of over-riding the inhibitory Ly-49G2 receptor blockade for cytokine production both in vitro and in vivo. This synergy is mediated by and dependent upon Ly-49D-expressing NK cells and results in significant systemic expression of IFN-gamma. This would place NK cells and their activating Ly-49 receptors as important initiators of microbial, antiviral, and antitumor immunity and provide a mechanism for the release of activating Ly-49 receptors from inhibitory receptor blockade. Little is known regarding the properties of activating Ly-49 NK receptors and their control of class I MHC-binding that has been shown to be important for recognition of tumors or virally infected host target cells. X-ray crystallography studies have identified two predominant contact sites (sites 1 and 2) that are involved in the binding of the inhibitory receptor, Ly-49A, to H-2D(d). Ly-49G2 (inhibitory) and Ly-49D (activating) are highly homologous to Ly-49A and also recognize H-2D(d). However, the binding of Ly-49D and G(2) to H-2D(d) is of lower affinity than Ly-49A. All Ly-49s contain N-glycosylation motifs; however, the importance of receptor glycosylation in Ly-49-class I interactions has not been determined. Ly-49D and G(2) contain a glycosylation motif (NTT (221-223)), absent in Ly-49A, adjacent to one of the proposed binding sites for H-2D(d) (site 2). The presence of a complex carbohydrate group at this critical site could interfere with class I binding. We are able to demonstrate for the first time that Ly-49D binds H-2D(d) in the presence of mouse beta(2)-microglobulin. We also demonstrate that glycosylation of the NTT (221-23) motif of Ly-49D interferes with recognition of H-2D(d). Alteration of the Ly-49D-NTT (221-23) motif to abolish glycosylation at this site resulted in enhanced H-2D(d) binding and receptor activation. Furthermore, glycosylation of Ly-49G2 at NTT (221-23) also reduces receptor binding to H-2D(d) tetramers. Therefore, the addition of complex carbohydrates to the Ly-49 family of receptors may represent a mechanism by which NK cells regulate affinity for host class I ligands. Thus we conclude that a primary role for the activating NK receptors in vivo is as a trigger for soluble factor production and regulation of the immune response at the site of receptor activation. This would place NK cells and their activating Ly-49 receptors as important initiators of microbial immunity and key elements of the innate immune system.